Registration Dossier

Diss Factsheets

Administrative data

Description of key information

The results obtained from an in vitro skin irritation test according to OECD guideline 439 and an in vitro skin corrosion test according to OECD guideline 431, using the EPISKIN model, indicate that the test item reveals no skin irritation or corrosion potential under the utilised testing conditions. In an in vitro eye corrosion test according to OECD guideline 438, the test item showed eye damaging effects.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Referenceopen allclose all

Endpoint:
skin corrosion: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
27-02-2020 to to 08-04-2020
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 431 (In Vitro Skin Corrosion: Reconstructed Human Epidermis (RHE) Test Method)
Version / remarks:
2019-06-14
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: Council Regulation (EC) No 440/2008, Annex Part B, B.40Bis: “In Vitro Skin Corrosion: Human Skin Model Test”, Official Journal of the European Union No. L142, dated May 31st, 2008.
Version / remarks:
2008-05-20
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: INVITTOX Protocol No. 118; “EPISKINTM Skin Corrosivity Test” (ECVAM Database Service on Alternative Methods to Animal Experimentation).
Version / remarks:
updated 2011-12 / 2012-02
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Test system:
human skin model
Source species:
other: reconstructed human epidermis
Cell type:
non-transformed keratinocytes
Justification for test system used:
The EPISKIN model has been validated for corrosivity testing in an international trial; it is considered to be suitable for this study (STATEMENT ON THE SCIENTIFIC VALIDITY OF THE EPISKINTM TEST (AN IN VITRO TEST FOR SKIN CORROSIVITY); ECVAM JRC Environment Institute, European Commission; Ispra; 03 April 1998).
Vehicle:
unchanged (no vehicle)
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiSkin SM, EPISKIN SNC Lyon, France, is a three-dimensional human epidermis model which develops a highly differentiated and stratified epidermis after a 13-day culture period.
- Tissue batch number: 20-EKIN-010

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: Room temperature

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: With 25 mL 1 x PBS solution, residual PBS was removed from the epidermal surface with a suitable pipette tip linked to a vacuum source (care was taken to avoid damaging to the epidermis)

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 0.3 mg/mL in assay medium, prepared from a 3 mg/mL stock solution (in 1x PBS), 2 mL per well
- Incubation time: 3 h (± 15 min)
- Wavelength: 570 nm

NUMBER OF REPLICATE TISSUES: 2

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
Approximately 50 μL test item was added to 2 mL MTT 0.3 mg/mL solution and mixed. The mixture was incubated for three hours at 37±1 °C, 5±1 % CO2 in a ≥ 95 % humidified atmosphere and protected from light. Subsequently, any observed colour changes of the solution was recorded. The test item did not interact with the MTT, therefore additional controls and data calculations were not necessary.

NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION: 1

PREDICTION MODEL / DECISION CRITERIA
- The test substance is considered to be corrosive to skin (Cat. 1A) if the viability after 3 minutes exposure is less than 35%.
- The test substance is considered to be corrosive to skin (Cat 1B and 1C) if the viability after 3 minutes exposure is greater than or equal to 35% and smaller than 35% after 60 minutes exposure; OR if the viability after 60 minutes exposure is greater than or equal to 35% and smaller than 35% after 240 minutes exposure.
- The test substance is considered to be non-corrosive to skin if the viability after 4 hours exposure is greater than or equal to 35%.
- Justification for the selection of the cut-off point: The cut-off value of 35 % and classification method was validated in an international validation of this kit (Fentem, 1998). The prediction model corresponds to the methods agreed by EU regulatory agencies in line with OECD 431 (OECD, 2015).
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
TEST MATERIAL
- Amount applied: 50 μL

NEGATIVE CONTROL
- Amount applied: 50 μL
- Concentration: 9 g/L

POSITIVE CONTROL
- Amount applied: 50 μL
Duration of treatment / exposure:
4 h
Number of replicates:
2
Irritation / corrosion parameter:
% tissue viability
Remarks:
mean of 2 replicates
Run / experiment:
1
Value:
64
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Other effects / acceptance of results:
- OTHER EFFECTS:
- Direct-MTT reduction: No colour change was observed after three hours of incubation. The test item did not interact with the MTT, therefore additional controls and data calculations were not necessary. A false estimation of viability can be precluded.
- Colour interference with MTT: The test item is colourless, furthermore showed no ability to become coloured in contact with water. Therefore, it was considered not to be able to significantly stain the tissues and lead to false estimate of viability. Additional controls and data calculations were not necessary. A false estimation of viability can be precluded.

DEMONSTRATION OF TECHNICAL PROFICIENCY: The laboratory demonstrated technical proficiency in a separate study using the twelve Proficiency Chemicals according to OECD Test Guideline No. 431.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes.
- Acceptance criteria met for positive control: Yes.
- Acceptance criteria met for variability between replicate measurements: Yes.

Table 1: Results of the optical density (OD) measured at 570 nm of each replicate and the calculated % viability of the cells (blank mean OD: 0.0386)





























































































Substance



Optical Density (OD)



Viability (%)



∆%



Negative control: NaCl (9 g/L)


4 h exposure



1



0.791



88



23.4



2



1.000



112



mean



0.895



100



SD



0.148



16.518



CV



16.518



16.518



Positive Control: Glacial acetic acid


4 h exposure



1



0.012



1



0.9



2



0.020



2



mean



0.016



2



SD



0.005



0.608



CV



34.754



34.754



Test item:


4 h exposure



1



0.571



64



0.7



2



0.577



64



mean



0.574



64



SD



0.004



0.466



CV



0.727



0.727



 Δ%: The difference of viability between the two relating tissues

Interpretation of results:
GHS criteria not met
Conclusions:
The results obtained from this in vitro skin corrosion test, using the EPISKIN model, indicated that the test item reveals no skin corrosion potential under the testing conditions. The test item can be classified as non-corrosive to skin.
Executive summary:

The EpiSkinTM test of the test item has been performed to predict its corrosion potential by measurement of its cytotoxic effect, as reflected in the MTT assay according to the OECD Test Guideline No. 431, adopted 14 June 2019. Disks of EPISKIN (two units / chemical / incubation time) were treated with test item and incubated for 4 hours (±10 min) at room temperature. Exposure of test material was terminated by rinsing with 1x PBS solution. The viability of each disk was assessed by incubating the tissues for 3 hours (±15 min) with MTT solution at 37±1 °C in an incubator with 5±1 % CO2 in a ≥ 95 % humidified atmosphere and protected from light. The formazan precipitated was then extracted using acidified isopropanol and quantified spectrophotometrically. NaCl (9 g/L saline) and glacial acetic acid treated epidermis were used as negative and positive controls, respectively. For each treated tissue viability was expressed as a % relative to negative control. The test item is considered to be non-corrosive to skin, if the mean relative viability after 4 hours of exposure is above or equal 35 % of the negative control. The test item did not show significantly reduced cell viability in comparison to the negative control after 4 hours of exposure. Both individual tissue viabilities were above 35 % of the mean negative control value. The average test item treated tissue viability was 64 %. Positive and negative controls showed the expected cell viability values within acceptable limits. All assay acceptance criteria were met, the experiment was considered to be valid.

Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2020-08-04 to 2020-08-28
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
other: EURL ECVAM DB-ALM Protocol n° 131
Version / remarks:
2012-06-09
Deviations:
no
Qualifier:
according to guideline
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Version / remarks:
2020-06-26
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)
Version / remarks:
2019-07-31
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: EpiSkin™ SOP, Version 1.8
Version / remarks:
February 2009
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Cell source:
other: adult human donors
Justification for test system used:
The EPISKIN model has been validated for irritation testing in an international trial. It showed evidence of being a reliable and relevant stand-alone test for predicting rabbit skin irritation, when the endpoint is evaluated by MTT reduction and for being used as a replacement for the Draize Skin Irritation test (OECD TG 404 and Method B.4 of Annex V to Directive 67/548/EEC) for the purposes of distinguishing between skin irritating and no skin irritating test substances (STATEMENT OF VALIDITY OF IN-VITRO TESTS FOR SKIN IRRITATION; ECVAM; Institute for Health & Consumer Protection; Joint Research Centre; European Commission; Ispra; 27 April 2007).
Vehicle:
unchanged (no vehicle)
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiSkin SM, EPISKIN SNC Lyon, France, is a three-dimensional human epidermis model which develops a highly differentiated and stratified epidermis after a 13-day culture period.
- Tissue batch number: 20-EKIN-032

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: Room temperature
- Temperature of post-treatment incubation: At 37 ± 1 °C in an incubator with 5 ± 1 % CO2, ≥ 95 % humidified atmosphere

REMOVAL OF TEST MATERIAL AND CONTROLS
- Volume and number of washing steps: With 25 mL 1 x PBS solution, residual PBS was removed from the epidermal surface with a suitable pipette tip linked to a vacuum source (care was taken to avoid damaging to the epidermis)

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 3 mg/mL stock solution; 2 mL of 0.3 mg/mL per well
- Incubation time: 3 hours
- Spectrophotometer: Thermo Scientific; Varioskan LUX
- Wavelength: 570 nm

NUMBER OF REPLICATE TISSUES: 3

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
Approximately 10 μL test item was added to 2 mL MTT 0.3 mg/mL solution and mixed. The mixture was incubated for three hours at 37±1 °C, 5±1 % CO2 in a ≥ 95 % humidified atmosphere and protected from light. Subsequently, any observed colour changes of the solution was recorded. The test item did not interact with the MTT, therefore additional controls and data calculations were not necessary.

NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION: 1

PREDICTION MODEL / DECISION CRITERIA
- The test substance is considered to be irritant to skin if the viability after 15 minutes exposure and 42 hours post incubation is less than or equal to 50% of the negative control.
- The test substance is considered to be non-irritant to skin if the viability after 15 minutes exposure and 42 hours post incubation is greater than 50% of the negative control.
Control samples:
yes, concurrent negative control
yes, concurrent positive control
yes, concurrent MTT non-specific colour control
Amount/concentration applied:
TEST MATERIAL
- Amount applied: 10 µL

VEHICLE
- Amount applied: 10 μL

POSITIVE CONTROL
- Amount applied: 10 μL
- Concentration: 5% aq. solution
Duration of treatment / exposure:
15 min
Duration of post-treatment incubation (if applicable):
42 hours
Number of replicates:
3
Irritation / corrosion parameter:
% tissue viability
Remarks:
mean of 3 replicates
Run / experiment:
1
Value:
79
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Other effects / acceptance of results:
- OTHER EFFECTS:
- Direct-MTT reduction: No colour change was observed after three hours of incubation. The test item did not interact with the MTT, therefore additional controls and data calculations were not necessary. A false estimation of viability can be precluded.
- Colour interference with MTT: The test item is colourless, furthermore showed no ability to become coloured in contact with water. Therefore, it was considered not to be able to significantly stain the tissues and lead to false estimate of viability. Additional controls and data calculations were not necessary. A false estimation of viability can be precluded.

DEMONSTRATION OF TECHNICAL PROFICIENCY:
Prior to routine use of the method, the test laboratory demonstrated the technical proficiency in a separate study using the ten Proficiency Chemicals according to OECD Test Guideline No. 439.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes.
- Acceptance criteria met for positive control: Yes.
- Acceptance criteria met for variability between replicate measurements: Yes.

Table 1: Results of the optical density (OD) measured at 570 nm of each replicate and the calculated % viability of the cells (blank mean OD: 0.040)






















































































Substance



Optical Density (OD)



Viability



Negative control: 1 x PBS



1



1.302



95



2



1.308



95



3



1.513



110



mean



1.375



100



standard deviation (SD)



8.72



Positive Control: SDS (5 % aq.)



1



0.095



7



2



0.086



6



3



0.125



9



mean



0.102



7



standard deviation (SD)



1.5



Test item:



1



1.081



79



2



1.182



86



3



0.999



73



mean



1.088



79



standard deviation (SD)



6.67



 

Interpretation of results:
GHS criteria not met
Conclusions:
The results obtained from this in vitro skin irritation test, using the EPISKIN model, indicated that the test item reveals no skin irritation potential under the utilised testing conditions. According to the current OECD Guideline No. 439, the test item is considered as non-irritant to skin and is therefore not classified (UN GHS No Category).
Executive summary:

The purpose of this study was to determine the skin irritation potential of the test item on reconstituted human epidermis in the EPISKIN model in vitro. Disks of epidermal units (three units) were treated with the test item and incubated for 15 ± 0.5 minutes at room temperature. Exposure of the test material was terminated by rinsing the epidermal units with 1x PBS solution. Epidermis units were then incubated at 37 ± 1 °C for 42 ± 1 hours (h) in an incubator with 5 ± 1 % CO2, ≥ 95 % humidified atmosphere. The viability of each disk was assessed by incubating the tissues for 3 hours ± 5 minutes with MTT solution at 37 ± 1 °C in 5 ± 1 % CO2, ≥ 95 % humidified atmosphere and protected from light. The resulting formazan crystals were extracted with acidified isopropanol and quantified by means of the optical densities (OD) recorded spectrophotometrically. SDS 5 % aq. and 1 x PBS treated epidermis units were used as positive and negative controls, respectively. For each treated tissue, viability was expressed as a percentage relative to negative control. The test item is identified as requiring classification and labelling according to UN GHS (Category 2 or Category 1), if the mean relative viability after 15 minutes exposure and 42 hours post incubation is less than or equal (≤) to 50 % when compared to the viability values obtained from the negative control. In this in vitro skin irritation test using the EPISKIN model, the test item 2-(allyloxy)ethanol did not show significantly reduced cell viability in comparison to the negative control (mean viability: 79 %). All obtained test item viability results were above 50 % when compared to the viability values obtained from the negative control. Therefore the test item was considered to be non-irritant to skin. Positive and negative controls showed the expected cell viability values within acceptable limits. All assay acceptance criteria were met, the experiment was considered to be valid.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records
Reference
Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2020-08-04 to 2020-09-04
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 438 (Isolated Chicken Eye Test Method for Identifying i) Chemicals Inducing Serious Eye Damage and ii) Chemicals Not Requiring Classification for Eye Irritation or Serious Eye Damage)
Version / remarks:
2018-06-25
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU method B.48 (Isolated chicken eye test method for identifying occular corrosives and severe irritants)
Version / remarks:
2017-02-14
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Species:
chicken
Details on test animals or tissues and environmental conditions:
SOURCE OF COLLECTED EYES
- Source: TARAVIS KFT. 9600 Sárvár, Rábasömjéni út 129. Hungary
- Characteristics of donor animals: Approximately 7 weeks old, 1.5 – 2.5 kg
- Storage, temperature and transport conditions of ocular tissue: Head collection was performed by a slaughter house technician. Heads were removed immediately after sedation of the chickens (sedation was happened by electric current). The heads were transported to Toxi-Coop ZRT. at the earliest convenience for use approximately within 2 hours from collection. The ambient temperature was optimal (19.3ºC to 20.4 ºC) during the transport. All eyes used in the assay were from the same groups of eyes collected on one specific day.
- Time interval prior to initiating testing: 2 h
- Indication of any existing defects or lesions in ocular tissue samples: After collection, the heads were inspected for appropriate quality. One small drop of fluorescein solution 2 % (w/v) was applied onto the cornea surface for a few seconds and subsequently rinsed off with 20 mL saline solution. Then the fluorescein-treated cornea was examined with a slit lamp microscope, with the eye in the head, to ensure that the cornea was not damaged (i.e., fluorescein retention ≤ 0.5).
Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
TEST MATERIAL
- Amount applied: 30 μL of test item was applied from micropipette onto the centre of the cornea.
Duration of treatment / exposure:
10 sec
Duration of post- treatment incubation (in vitro):
After an exposure period of 10 seconds, the cornea surface was rinsed thoroughly with 20 mL isotonic saline. The time while the eye was out of the chamber was limited to a minimum.
Number of animals or in vitro replicates:
3 eyes each (positive control +test item) and 1 eye (negative control)
Details on study design:
SELECTION AND PREPARATION OF ISOLATED EYES
Heads were inspected for appropriate quality and wrapped with paper moistened with saline, then placed in a plastic box. After removing the head from the plastic box, it was put on soft paper. The eyelids were carefully cut away with scissors, avoiding damaging the cornea. One small drop of fluorescein solution 2 % (w/v) was applied onto the cornea surface for a few seconds and subsequently rinsed off with 20 mL saline solution. Only corneas in good condition (i.e., fluorescein retention ≤ 0.5) were used. The eyeball was carefully removed from the orbit by holding the nictitating membrane with a surgical forceps, while cutting the eye muscles with bent scissors. Care was taken to remove the eyeball from the orbit without cutting off the optical nerve too short. The procedure avoided pressure on the eye while removing the eyeball from the orbit, in order to prevent distortion of the cornea and subsequent corneal opacity. Once removed from the orbit, the eye was placed onto damp paper. The nictitating membrane and other connective tissue were cut away. The prepared eyes were kept on wet papers in a closed box to maintain an appropriate humidity. The treatment group and the concurrent positive control consisted of three eyes. The negative control group consisted of one eye. The enucleated eye was placed in a steel clamp with the cornea positioned vertically with the eye in the correct relative position (same position as in the chicken head). Again, too much pressure on the eye by the clamp was avoided. Because of the relatively firm sclera of the chicken eyeball, only slight pressure was needed to fix the eye properly. The clamp with the eyeball was transferred to a chamber of the superfusion apparatus. The clamp holding the eye was positioned in such a way that the entire cornea was supplied with saline solution dripping from a stainless steel tube, at a rate of approximately 3 or 4 drops/minute. The door of the chamber was closed except for manipulations and examinations, to maintain temperature and humidity. The appropriate number of eyes was selected and, after being placed in the superfusion apparatus, the eyes were examined again with the slit lamp microscope to ensure that they were in good condition. The focus was adjusted to see clearly the saline solution which was flowing on the cornea surface. Eyes with a high baseline fluorescein staining (i.e., > 0.5) or a high corneal opacity score (i.e., > 0.5) were rejected. The cornea thickness was measured using the depth measuring device. Any eye with cornea thickness deviating more than 10 % from the mean value for the eyes, or eyes that showed any other signs of damage, were rejected and replaced. If the selected eyes were appropriate for the test, acclimatisation started and was conducted for approximately 45 to 60 minutes. The temperature of the circulating water was verified to ensure that the temperature in all chambers was in the range of 32 ± 1.5 °C during the acclimatisation and treatment periods.

EQUILIBRATION AND BASELINE RECORDINGS
At the end of the acclimatization period, a zero reference measurement was recorded for cornea thickness and opacity to serve as a baseline (t=0) for each individual eye. The cornea thickness of the eyes should not change by more than ±5-7 % within approximately 45 to 60 minutes before the start of application. Slight changes in thickness (0% to 3%) were observed in the eyes, finding considered as normal when maintaining enucleated eyes. Following the equilibration period, the fluoresce in retention was measured. Baseline values were required to evaluate any potential test item related effects after treatment. The location of any minor findings was marked on the record sheet as a drawing, if applicable. If any eye was considered to be unsuitable following baseline assessment, it was discarded.

NUMBER OF REPLICATES: 3 eyes each (positive control +test item) and 1 eye (negative control)

NEGATIVE CONTROL USED: NaCl (9 g/L saline)

POSITIVE CONTROL USED: Acetic acid 10 % (v/v)

APPLICATION DOSE AND EXPOSURE TIME: 30 µL for 10 sec

OBSERVATION PERIOD: The control- and test item-treated eyes were evaluated pre-treatment and at approximately 30, 75, 120, 180 and 240 minutes after the post-treatment rinse. The cornea thickness was measured and cornea opacity and possible morphological effects were observed at all time points. Fluorescein retention was measured on two occasions, at base line (t=0) and 30 minutes after the post-treatment rinse.

REMOVAL OF TEST SUBSTANCE
- Volume and washing procedure after exposure period: After an exposure period of 10 seconds, the cornea surface was rinsed thoroughly with 20 mL saline solution at ambient temperature, while taking care not to damage the cornea but attempting to remove the entire residual test item, if possible. The eye in the holder was then returned to its chamber. The time while the eye was out of the chamber was limited to a minimum.

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: Examination with a slit lamp microscope
- Damage to epithelium based on fluorescein retention: By a small drop of fluorescein solution 2 % (w/v) applied onto the cornea surface for a few seconds and subsequently rinsed off with 20 mL saline solution. Then the fluorescein-treated cornea was examined with a slit lamp microscope.
- Swelling: Measured with a depth measuring device on the slit lamp microscope (Haag-Streit BQ 900) with the slit-width set at 9½, equaling 0.095 mm.

SCORING SYSTEM:
- Mean corneal swelling (%)
- Mean maximum opacity score
- Mean fluorescein retention score at 30 minutes post-treatment

DECISION CRITERIA: Decision criteria as indicated in the TG were used.
Irritation parameter:
percent corneal swelling
Run / experiment:
1 (mean of 3 eyes)
Value:
27
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
positive indication of irritation
Irritation parameter:
cornea opacity score
Run / experiment:
1 (mean of 3 eyes)
Value:
3.7
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
positive indication of irritation
Irritation parameter:
fluorescein retention score
Run / experiment:
1 (mean of 3 eyes)
Value:
3
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
positive indication of irritation
Other effects / acceptance of results:
OTHER EFFECTS:
- Visible damage on test system: No.

DEMONSTRATION OF TECHNICAL PROFICIENCY: Prior to routine use of the method, the testing facility demonstrated the technical proficiency in a separate study using the ten Proficiency Chemicals according to OECD Test Guideline No. 438.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes.
- Acceptance criteria met for positive control: Yes.
Interpretation of results:
Category 1 (irreversible effects on the eye) based on GHS criteria
Conclusions:
In this ICET, the test item caused ocular corrosion or severe irritation in the enucleated chicken eye.
Executive summary:

The purpose of this Isolated Chicken Eye Test (ICET) was to evaluate the potential ocular corrosivity or severe irritancy of the test item by its ability to induce toxicity in enucleated chicken eyes. The test compound was applied in a single dose onto the cornea of isolated chicken eyes in order to potentially classify the test compound as either 1: causing "serious eye damage" [category 1 of the Globally Harmonised System for the Classification and Labelling of chemicals (GHS)], or not requiring classification for eye irritation or serious eye damage according to the GHS. The ICET does not fully replace the in vivo rabbit eye test (OECD 405); however, the ICET is used as part of a tiered testing strategy for regulatory purposes. The test item, acetic acid 10 % (v/v) solution (positive control) and NaCl (9 g/L saline) (negative control) were applied in such a way that the entire surface of the cornea was uniformly covered with the test item or positive or negative control. Three test item treated eyes and three positive control eyes and one negative control eye were used in this study. After an exposure period of 10 seconds, the cornea surface was rinsed thoroughly with ~20 mL saline solution at ambient temperature and this procedure was repeated for each eye. In this ICET, the test item caused ocular corrosion or severe irritation in the enucleated chicken eye. The overall ICE classes were once III (based on corneal swelling of 27 % within 240 min) and twice IV (based on corneal opacity score of 3.7 and fluorescein retention score of 3.0). The positive control was classified as corrosive/severely irritating, UN GHS Classification: Category 1 and the negative control had no significant effects on the chicken eye in this study. Furthermore, the three endpoints of the positive and the negative controls were in the historical control range. So the positive and negative controls showed the expected results. The experiment was considered to be valid in both occasions.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (irreversible damage)

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Skin corrosion, key study


The EpiSkin test of the test item has been performed to predict its corrosion potential by measurement of its cytotoxic effect, as reflected in the MTT assay according to the OECD Test Guideline No. 431, adopted 14 June 2019. Disks of EPISKIN (two units / chemical / incubation time) were treated with test item and incubated for 4 hours (±10 min) at room temperature. Exposure of test material was terminated by rinsing with 1x PBS solution. The viability of each disk was assessed by incubating the tissues for 3 hours (±15 min) with MTT solution at 37±1 °C in an incubator with 5±1 % CO2 in a ≥ 95 % humidified atmosphere and protected from light. The formazan precipitated was then extracted using acidified isopropanol and quantified spectrophotometrically. NaCl (9 g/L saline) and glacial acetic acid treated epidermis were used as negative and positive controls, respectively. For each treated tissue viability was expressed as a % relative to negative control. The test item is considered to be non-corrosive to skin, if the mean relative viability after 4 hours of exposure is above or equal 35 % of the negative control. The test item did not show significantly reduced cell viability in comparison to the negative control after 4 hours of exposure. Both individual tissue viabilities were above 35 % of the mean negative control value. The average test item treated tissue viability was 64 %. Positive and negative controls showed the expected cell viability values within acceptable limits. All assay acceptance criteria were met, the experiment was considered to be valid.


 


The EPISKIN test method according to OECD guideline 431 is designed to detect skin damaging substance properties. Irritating effects cannot be excluded by this method, therefore a test for skin irritation was performed in a second step.


 


Skin irritation, key study


The purpose of this study was to determine the skin irritation potential of the test item on reconstituted human epidermis in the EPISKIN model in vitro according to OECD guideline 439. Disks of epidermal units (three units) were treated with the test item and incubated for 15 ± 0.5 minutes at room temperature. Exposure of the test material was terminated by rinsing the epidermal units with 1x PBS solution. Epidermis units were then incubated at 37 ± 1 °C for 42 ± 1 hours (h) in an incubator with 5 ± 1 % CO2, ≥ 95 % humidified atmosphere. The viability of each disk was assessed by incubating the tissues for 3 hours ± 5 minutes with MTT solution at 37 ± 1 °C in 5 ± 1 % CO2, ≥ 95 % humidified atmosphere and protected from light. The resulting formazan crystals were extracted with acidified isopropanol and quantified by means of the optical densities (OD) recorded spectrophotometrically. SDS 5 % aq. and 1 x PBS treated epidermis units were used as positive and negative controls, respectively. For each treated tissue, viability was expressed as a percentage relative to negative control. The test item is identified as requiring classification and labelling according to UN GHS (Category 2 or Category 1), if the mean relative viability after 15 minutes exposure and 42 hours post incubation is less than or equal (≤) to 50 % when compared to the viability values obtained from the negative control. In this in vitro skin irritation test using the EPISKIN model, the test item 2-(allyloxy)ethanol did not show significantly reduced cell viability in comparison to the negative control (mean viability: 79 %). All obtained test item viability results were above 50 % when compared to the viability values obtained from the negative control. Therefore the test item was considered to be non-irritant to skin. Positive and negative controls showed the expected cell viability values within acceptable limits. All assay acceptance criteria were met, the experiment was considered to be valid.


 


QSAR, supporting study


The Danish QSAR models were used for prediction of skin irritation or corrosion properties. The test item was determined not to be irritating or corrosive to skin based on the described QSAR method.


 


The adequacy of a prediction depends on the following conditions:



  1. a) the (Q)SAR model is scientifically valid: the scientific validity is established according to the OECD principles for (Q)SAR validation;

  2. b) the (Q)SAR model is applicable to the query chemical: a (Q)SAR is applicable if the query chemical falls within the defined applicability domain of the model;

  3. c) the (Q)SAR result is reliable: a valid (Q)SAR that is applied to a chemical falling within its applicability domain provides a reliable result;

  4. d) the (Q)SAR model is relevant for the regulatory purpose.


 


For assessment and justification of these 4 requirements the QMRF and QPRF files were developed and attached to this study record.


 


Description of the prediction Model


The prediction model was descripted using the harmonised template for summarising and reporting key information on (Q)SAR models. For more details please refer to the attached QSAR Model Reporting Format (QMRF) file. 


 


Assessment of estimation domain


The assessment of the estimation domain was documented in the QSAR Prediction Reporting Format file (QPRF). Please refer to the attached document for the details of the prediction and the assessment of the estimation domain.


 


Conclusion: In summary, it can be concluded that the test substance has no skin irritating or corrosive properties.


 


Eye corrosion, key study


The purpose of this Isolated Chicken Eye Test (ICET) was to evaluate the potential ocular corrosivity or severe irritancy of the test item by its ability to induce toxicity in enucleated chicken eyes. The test compound was applied in a single dose onto the cornea of isolated chicken eyes in order to potentially classify the test compound as either 1: causing "serious eye damage" [category 1 of the Globally Harmonised System for the Classification and Labelling of chemicals (GHS)], or not requiring classification for eye irritation or serious eye damage according to the GHS. The ICET does not fully replace the in vivo rabbit eye test (OECD 405); however, the ICET is used as part of a tiered testing strategy for regulatory purposes. The test item, acetic acid 10 % (v/v) solution (positive control) and NaCl (9 g/L saline) (negative control) were applied in such a way that the entire surface of the cornea was uniformly covered with the test item or positive or negative control. Three test item treated eyes and three positive control eyes and one negative control eye were used in this study. After an exposure period of 10 seconds, the cornea surface was rinsed thoroughly with ~20 mL saline solution at ambient temperature and this procedure was repeated for each eye. In this ICET, the test item caused ocular corrosion or severe irritation in the enucleated chicken eye. The overall ICE classes were once III (based on corneal swelling of 27 % within 240 min) and twice IV (based on corneal opacity score of 3.7 and fluorescein retention score of 3.0). The positive control was classified as corrosive/severely irritating, UN GHS Classification: Category 1 and the negative control had no significant effects on the chicken eye in this study. Furthermore, the three endpoints of the positive and the negative controls were in the historical control range. So the positive and negative controls showed the expected results. The experiment was considered to be valid in both occasions.

Justification for classification or non-classification

Classification, Labeling, and Packaging Regulation (EC) No 1272/2008


The available test data are reliable and suitable for classification purposes under Regulation (EC) No 1272/2008. Thus, the test item is considered not to be classified for skin irritation/corrosion, but for serious eye damage (category 1) under Regulation (EC) No 1272/2008, as amended for fifteenth time in Regulation (EU) No 2020/1182.